Apoptosis and exposure of phosphatidylserine on the cell surface is a common feature of programmed cell death from species ranging from invertebrates such as C. elegans and D. melanogaster through vertebrate phyla to humans. The breakdown of the asymmetry of membrane phospholipids and increased exposure of phosphatidylserine on the cell surface leads to enhanced annexin V binding and provides a signal for phagocytosis by mononuclear cells. It has recently been reported that phosphatidylserine exposure during apoptosis reflects bi-directional trafficking of a particular subset of membrane vesicles between the cell surface and the cytoplasm.
Apoptosis is commonly characterized by condensation of nuclear chromatin, and fragmentation of nuclear structure into so-called apoptotic bodies. At present, there exists a variety of techniques that can detect the process of apoptosis at different stages. For example, the terminal stage of apoptosis can be assayed by morphological changes of the cell (such as the presence of apoptotic bodies). Before that, apoptosis can be assayed by DNA fragmentation using either gel analysis or the TUNEL technique. Earlier stages of apoptosis can be assayed by exposure of phosphatidylserine in the outer sheath of the membrane using an annexin V labelled protein, or by detecting the activation of caspase-3 using a fluorescent dye linking to a substrate peptide.
All of these techniques, however, have certain limitations. For example, gel analysis can only be applied to an extract of cells, not to a single cell or intact cells. The TUNEL method can only be applied to fixed cells, not living cells. annexin V can only detect events at the outer cell surface, not inside the cell. The caspase probe using a peptide linked fluorescence dye also has limitations. First, this probe cannot penetrate the cell membrane, and thus, it is typically used to assay cell extract. Secondly, the fluorescent change resulting from caspase cleavage involves mainly a shift of the emission spectrum in the dye rather than a total destruction of the fluorescence, and sensitivity is limited.
The most popular method for detecting apoptosis is the molecular recognition of surface-exposed phosphatidylserine that is based on the ability of annexin V to interact with phosphatidylserine exposed on the surface in a Ca2+-dependent manner. Different variants of this method have been developed. For example, annexin V labelled with fluorescein has been used for flow cytometry, while annexin V labelled with red-near infrared dyes has been used for tissue imaging. This protein was also labelled with colloid gold for electron microscopy, with radioactive tracer for autoradiography on the tissue level and with peroxidase for histochemical studies. In all these tests, a high (up to 2.5 mM) extracellular concentration of Ca2+ ions has to be provided for complete binding of annexin V to phosphatidylserine. For this reason, special buffers are required in order to detect surface phosphatidylserine. Furthermore, annexin V can associate with membrane surfaces containing by-products of lipid per oxidation that modify amines by producing negative charges, and detergents in the medium can also change the annexin V lipid binding specificity. In addition, routinely used cell harvesting techniques for adhering cells, such as trypsinization, can also produce false results in application of this method.
Thus, there remains a need for methods and compositions for the detection of cells undergoing cell death.